Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices

Separations of 15 pM rhodamine 6G and 30 pM rhodamine B performed in a micromachined electrophoresis channel were detected by counting fluorescence bursts from individual molecules. The migration times, peak widths, and analyte concentrations were estimated from the number and the migration time distribution of the detected molecules. Concentration detection limits estimated at >99% confidence were 1.7 pM rhodamine 6G and 8.5 pM rhodamine B. The separations required <35 s and the relative migration time uncertainties were less than 2.0%. These are the lowest detection limits reported for microchip separation devices and the first example of single-chromophore molecular counting for detection of chemical separations.